scholarly journals Lactobacillus Strain Diversity Based on Partial hsp60 Gene Sequences and Design of PCR-Restriction Fragment Length Polymorphism Assays for Species Identification and Differentiation

2007 ◽  
Vol 74 (1) ◽  
pp. 208-215 ◽  
Author(s):  
Giuseppe Blaiotta ◽  
Vincenzina Fusco ◽  
Danilo Ercolini ◽  
Maria Aponte ◽  
Olimpia Pepe ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Direct Sequencing ◽  
Nucleotide Sequences ◽  
Gene Sequences ◽  
Restriction Fragment Length

ABSTRACT A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.

scholarly journals Analysis of the Endophytic Actinobacterial Population in the Roots of Wheat (Triticum aestivum L.) by Terminal Restriction Fragment Length Polymorphism and Sequencing of 16S rRNA Clones

2004 ◽  
Vol 70 (3) ◽  
pp. 1787-1794 ◽  
Author(s):  
Vanessa M. Conn ◽  
Christopher M. M. Franco
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Restriction Fragment Length

ABSTRACT The endophytic actinobacterial population in the roots of wheat grown in three different soils obtained from the southeast part of South Australia was investigated by terminal restriction fragment length polymorphism (T-RFLP) analysis of the amplified 16S rRNA genes. A new, validated approach was applied to the T-RFLP analysis in order to estimate, to the genus level, the actinobacterial population that was identified. Actinobacterium-biased primers were used together with three restriction enzymes to obtain terminal restriction fragments (TRFs). The TRFs were matched to bacterial genera by the T-RFLP Analysis Program, and the data were analyzed to validate and semiquantify the genera present within the plant roots. The highest diversity and level of endophytic colonization were found in the roots of wheat grown in a dark loam from Swedes Flat, and the lowest were found in water-repellent sand from Western Flat. This molecular approach detected a greater diversity of actinobacteria than did previous culture-dependent methods, with the predominant genera being Mycobacterium (21.02%) in Swedes Flat, Streptomyces (14.35%) in Red Loam, and Kitasatospora (15.02%) in Western Flat. This study indicates that the soil that supported a higher number of indigenous organisms resulted in wheat roots with higher actinobacterial diversity and levels of colonization within the plant tissue. Sequencing of 16S rRNA clones, obtained using the same actinobacterium-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity and identified a number of Mycobacterium and Streptomyces species.

scholarly journals Detection and Characterization of a Dehalogenating Microorganism by Terminal Restriction Fragment Length Polymorphism Fingerprinting of 16S rRNA in a Sulfidogenic, 2-Bromophenol-Utilizing Enrichment

2004 ◽  
Vol 70 (2) ◽  
pp. 1169-1175 ◽  
Author(s):  
Donna E. Fennell ◽  
Sung-Keun Rhee ◽  
Young-Beom Ahn ◽  
Max M. Häggblom ◽  
Lee J. Kerkhof
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Reductive Dehalogenation ◽  
Polymorphism Analysis ◽  
Sulfate Reducing ◽  
Restriction Fragment Length

ABSTRACT Terminal restriction fragment length polymorphism analysis of reverse-transcribed 16S rRNA during periods of community flux was used as a tool to delineate the roles of the members of a 2-bromophenol-degrading, sulfate-reducing consortium. Starved, washed cultures were amended with 2-bromophenol plus sulfate, 2-bromophenol plus hydrogen, phenol plus sulfate, or phenol with no electron acceptor and were monitored for substrate use. In the presence of sulfate, 2-bromophenol and phenol were completely degraded. In the absence of sulfate, 2-bromophenol was dehalogenated and phenol accumulated. Direct terminal restriction fragment length polymorphism fingerprinting of the 16S rRNA in the various subcultures indicated that phylotype 2BP-48 (a Desulfovibrio-like sequence) was responsible for the dehalogenation of 2-bromophenol. A stable coculture was established which contained predominantly 2BP-48 and a second Desulfovibrio-like bacterium (designated BP212 based on terminal restriction fragment length polymorphism fingerprinting) that was capable of dehalogenating 2-bromophenol to phenol. Strain 2BP-48 in the coculture could couple reductive dehalogenation to growth with 2-bromophenol, 2,6-dibromophenol, or 2-iodophenol and lactate or formate as the electron donor. In addition to halophenols, strain 2BP-48 appears to use sulfate, sulfite, and thiosulfate as electron acceptors and is capable of simultaneous sulfidogenesis and reductive dehalogenation in the presence of sulfate.

scholarly journals Application of New Primer-Enzyme Combinations to Terminal Restriction Fragment Length Polymorphism Profiling of Bacterial Populations in Human Feces

2003 ◽  
Vol 69 (2) ◽  
pp. 1251-1262 ◽  
Author(s):  
Koji Nagashima ◽  
Takayoshi Hisada ◽  
Maremi Sato ◽  
Jun Mochizuki
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rrna Gene ◽  
The 16S Rrna Gene ◽  
Restriction Fragment Length

ABSTRACT New primer-enzyme combinations for terminal restriction fragment length polymorphism (T-RFLP) targeting of the 16S rRNA gene were constructed by using the T-RFLP analysis program (designated TAP T-RFLP) located at the Ribosomal Database Project website, and their performance was examined empirically. By using the fluorescently labeled 516f primer (Escherichia coli positions 516 to 532) and 1510r primer (positions 1510 to 1492), the 16S rRNA gene was amplified from human fecal DNA. The resulting amplified product was digested with RsaI plus BfaI or with BslI. When the T-RFLP was carried out with fecal DNAs from eight individuals, eight predominant operational taxonomic units (OTUs) were detected with RsaI and BfaI digestion and 14 predominant OTUs were detected with BslI digestion. The distribution of the OTUs was consistent with the results of the computer simulations with TAP T-RFLP. The T-RFLP analyses of the fecal DNAs from individuals gave characteristic profiles, while the variability of the T-RFLP profiles between duplicate DNA preparations from the same samples were minimal. This new T-RFLP method made it easy to predict what kind of intestinal bacterial group corresponded to each OTU on the basis of the terminal restriction fragment length compared with the conventional T-RFLP and, moreover, made it possible to identify the bacterial species that an OTU represents by cloning and sequencing.

scholarly journals KARAKTERISASI POPULASI IKAN PUTAK (Notopterus notopterus) MENGGUNAKAN ANALISIS KERAGAMAN FENOTIPIK DAN DAERAH 16 SRNA DNA MITOKONDRIA

2017 ◽  
Vol 15 (1) ◽  
pp. 1
Author(s):  
Arif Wibowo ◽  
Mas Tri Djoko Sunarno ◽  
Subagdja Subagdja ◽  
Taufiq Hidayah
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Morphological Characters ◽  
Purposive Sampling ◽  
Notopterus Notopterus ◽  
Restriction Fragment Length

Penelitian ini dilakukan pada tahun 2006 di Sungai Ogan, Sungai Kelekar (anak Sungai Musi, Sumatera Selatan), perairan di Pulau Bangka serta Kota Bangun dan Tanah Ulu (Sungai Mahakam, Kalimantan Timur). Tujuannya adalah untuk menganalisis karakterisasi populasi ikan putak (Notopterus notopterus) dari berbagai habitat berdasarkan pada karakter fenotipik dan genetik serta untuk menentukan karakter morfologi sebagai pembeda utama, mendeterminasi jarak kemiripan antar populasi ikan putak dan untuk menganalisis apakah perbedaan morfologi antar populasi tersebut hanya sekedar fenotipik plastisity atau memiliki dasar evolusi yang signifikan. Dari lokasi pengambilan contoh yang ditentukan secara sengaja (purposive sampling) berhasil dikoleksi 49 spesimen untuk pengukuran morfometrik dan meristik yang berasal dari lima lokasi berbeda dan tujuh spesimen dari dua populasi untuk marka molekuler. Pengukuran biometrik dilakukan pada 35 karakter morfologi bentuk badan pada bagian sisi sebelah kiri tubuh ikan. Data biometrik dianalisis dengan Analisis Deskriminan, menggunakan Software Statistica 6.0, sedangkan data genetik menggunakan Metode Restriction Fragment Length Polymorphism (RFLP) pada mitokondria (mtDNA), menggunakan primer 16S rRNA, hasilnya dianalis secara manual kualitatif. Hasil penelitian menunjukkan bahwa berdasarkan pada analisis karakter morfologi, populasi ikan putak yang terdapat di Sungai Ogan, Sungai Kelekar, perairan di Bangka, dan Sungai Mahakam (Kota Bangun dan Tanah Ulu) merupakan populasi yang terpisah. Karakter pembeda utama antar kelompok populasi tersebut adalah Snouth Length (SL), Isthmus Length (IL), dan Adipose Length (AL). Populasi ikan putak yang berada di Pulau Kalimantan (Tanah Ulu dan Kota Bangun) memiliki jarak kemiripan yang cukup jauh dengan populasi di Pulau Sumatera (Ogan, Kelekar dan Bangka). Terlihat jelas bahwa pola isolasi dikarenakan oleh barrier alam. Perbedaan antar populasi ikan putak tidak hanya merupakan plastisity fenotipik, namun juga telah memiliki dasar evolusi. This reseach was conducted at 2006 in the rivers of Ogan and Kelekar (segment of Musi River, South Sumatera), waters in Bangka Island, and Kota Bangun, and Tanah Ulu (segment of Mahakam River, East Kalimantan). This study was to reveal population characteristic of putak (Notopterus notopterus) from different habitats based on its characters of fenotipic and genetic, and to determine the most discriminate morphological characters, to determine similiarty distance among the putak population and to analyze whether morphological differences among those populations are just phenotipic plastisity or it has evolution significantly. Of sampling locations selected using purposive sampling were got 49 specimens for measurement of morphometric and meristic originated from five different locations and seven specimens for mtDNA analysis originated from two different populations. Biometric measurement was conducted at 35 morphological characters on the leftside of the fish body. Restriction Fragment Length Polymorphism(RLFP)Method was employed formt DNA analysis using 16S rRNA marker. Biometric datum was subject to Discriminant Analysis using Statistica 6.0 package, since mtDNA was analyzed by manual qualitative. The results on morphological analysis showed that the putak originated from Ogan River, Kelekar, inland waters of Bangka Island, Kota Bangun, and Tanah Ulu were separated population. Snouth Length (SL), Isthmus Length (IL), and Adipose Length (AL) are the most discriminate morphological characters. Population of the putak from Kalimantan Island (Tanah Ulu and Kota Bangun) had wider similarity distance compared to from that Sumatera population (Ogan River, Kelekar River, and Bangka Island). It was clearly shown that  the putak population among different islands was isolated by natural barrier. Morphological difference of the putak was not only caused by fenotipic, but also by evolution significance.

scholarly journals 16S rRNA Terminal Restriction Fragment Length Polymorphism for the Characterization of the Nasopharyngeal Microbiota

PLoS ONE ◽  
2012 ◽  
Vol 7 (12) ◽  
pp. e52241 ◽  
Author(s):  
Silvio D. Brugger ◽  
Laurence Frei ◽  
Pascal M. Frey ◽  
Suzanne Aebi ◽  
Kathrin Mühlemann ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rrna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Restriction Fragment Length
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